EP1496111A2 - Variants of 3-Phosphoglycerate dehydrogenase with reduced inhibition by L-serine and genes encoding the same - Google Patents
Variants of 3-Phosphoglycerate dehydrogenase with reduced inhibition by L-serine and genes encoding the same Download PDFInfo
- Publication number
- EP1496111A2 EP1496111A2 EP04015524A EP04015524A EP1496111A2 EP 1496111 A2 EP1496111 A2 EP 1496111A2 EP 04015524 A EP04015524 A EP 04015524A EP 04015524 A EP04015524 A EP 04015524A EP 1496111 A2 EP1496111 A2 EP 1496111A2
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- EP
- European Patent Office
- Prior art keywords
- pgd
- amino acid
- sera
- amino acids
- codon
- Prior art date
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Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01095—Phosphoglycerate dehydrogenase (1.1.1.95)
Definitions
- the invention relates to variants of 3-phosphoglycerate dehydrogenase with reduced inhibition by L-serine and coding for it Genes.
- biosynthetic pathways are subject to wild-type strains a strict control that ensures that the amino acids be made only for personal use of the cell.
- important control mechanism in many biosyntheses the phenomenon of feedback inhibition (or end product inhibition).
- the enzyme of a biosynthetic pathway this is the preliminary enzymatic reaction of this Biosynthesis pathways catalyzed by the end product of Biosyntheseweges inhibited.
- the inhibition usually takes place through an allosteric Binding of the final product to the enzyme instead of and causes a conformational change to an inactive state. This ensures that when an accumulation of the final product in the cell further synthesis by inhibition of introductory step is prevented.
- the phosphoglycerate family of amino acids is defined by that they are amino acids involved in their biosynthesis derived from the 3-phosphoglyceric acid. Of the natural path of metabolism leads to it over the intermediates 3-Phospohydroxypyruvat and 3-phospho-Lserin to L-serine. L-serine can continue to glycine or over O-acetyl-serine be converted to L-cysteine. Also L-tryptophan is to be counted to this group, since it is also in derived from the biosynthesis of L-serine. Likewise, unnatural Amino acids described in US 2002/0039767 A1 Process to be assigned to the phosphoglycerate family.
- the initial step in the biosynthesis of amino acids of the phosphoglycerate family is the oxidation of D-3-phosphoglyceric acid to 3-phosphohydroxypyruvate and is catalyzed by the enzyme 3-phosphoglycerate dehydrogenase (PGD) [EC 1.1.1.95].
- PGD 3-phosphoglycerate dehydrogenase
- NAD + which is converted into NADH / H + , serves as the acceptor for the reduction equivalents formed during the reaction.
- PGD enzymes are known from a variety of organisms (e.g. Rattus norvegicus, Arabidopsis thaliana, Escherichia coli, Bacillus subtilis). The better characterized microbial representatives of feedback inhibition by L-serine.
- amino acid 1-340 of the E. coli sequence amino acid 1-340 of the E. coli sequence
- C-terminal portion has little similarity.
- the C-terminal Part is localized to the regulatory domain, the is responsible for serine inhibition (Peters-Wendisch et al., 2002, Appl. Microbiol. Biotechnol. 60: 437-441).
- PGD The best characterized PGD is that of Escherichia coli.
- the enzyme has been extensively biochemically studied (Dubrow & Pizer, 1977, J. Biol. Chem. 252, 1527-1538) and is subject to allosteric feedback inhibition by L-serine with an inhibitor constant K i of 5 ⁇ M.
- linker mutagenesis are usually problematic because by the incorporation or deletion of several residues the Structure of the protein is very much changed and so the overall activity or stability of the protein adversely affected becomes. In fact, most of them are described in EP0620853A Mutants a barely detectable activity.
- Object of the present invention was to variants of PGD of Escherichia coli which have an im Compared to Escherichia coli wild-type PGD decreased sensitivity against inhibition by serine.
- a PGD having an amino acid sequence which is characterized by being different from the Amino acid sequence of Escherichia coli wild-type PGD (SEQ ID NO: 2) with a methionine as position 1 differs thereby that at position 349 an amino acid except glycine or at position 372 an amino acid except threonine located.
- a PGD according to the invention may also have mutations in both Have positions of SEQ ID NO: 1.
- the invention further relates to a DNA sequence coding for a PGD according to the invention.
- This serA allele is different different from the gene of Escherichia coli PGD (serA gene, SEQ ID NO: 1) in that the codon 349 is a natural amino acid Exception of glycine or codon 372 for a natural amino acid coded with the exception of threonine.
- a serA allele according to the invention may also have a mutation have two codons mentioned.
- inventive serA alleles include such genes that are analyzed with the algorithm GAP (GCG Wisconsin Package, Genetics Computer Group (GCG) Madison, Wisconsin) a sequence identity greater than 30% if they have any of the above mutations exhibit. Particularly preferred is a sequence identity greater than 70%.
- proteins with a sequence identity greater than 40 % determined with the algorithm GAP in the sense of the present To conceive of the invention as proteins derived from E. coli PGD, provided they have a PGD activity and one of the above Have amino acid substitutions. Particularly preferred is a Sequence identity greater than 70%.
- genes according to the invention are allelic variants of the serA gene to be understood by deletion, insertion or Substitution of nucleotides from that shown in SEQ ID NO: 1 Derive sequence, with the enzymatic activity of the gene product more than 10% of the activity of the wild-type gene product and a mutation of codon 349 for the Amino acid glycine or codon 372 for the amino acid threonine, or a combination of the two mutations.
- PGD variants that have an amino acid substitution at position 349 or at position 372 or a combination thereof, can be generated using standard techniques of molecular biology become. For this purpose, mutations are made in the corresponding codons introduced the serA gene encoding the PGD. Appropriate Methods for introducing mutations at specific positions within a DNA fragment are known.
- the starting material for the mutagenesis is preferably the DNA of the E. coli serA gene.
- the serA gene to be mutated can chromosomally or extrachromosomally encoded. It is preferred However, the serA gene amplified by polymerase chain reaction and cloned into a vector.
- the DNA sequence is altered so that the encoded PGD undergoes an amino acid exchange at position 349 or 372, wherein position 1 is the starting methionine of SEQ ID NO: 1.
- any method can be used which allows to determine the activity of the enzyme in the presence of L-serine.
- the determination of PGD activity can be based on the method described by McKrickick and Pizer (1980, J. Bacteriol 141: 235-245).
- the enzyme activity is measured by the reverse reaction in an approach containing phosphohydroxypyruvate and NADH / H + .
- the reaction is started by enzyme addition and monitored by the decrease in absorbance at 340 nm, which is caused by oxidation of NADH / H + in a spectrophotometer.
- the inhibition of PGD activity is tested in the presence of various concentrations of L-serine in the reaction mixture.
- the catalytic activity of the various PGD variants is determined in the presence and absence of L-serine and used to determine the inhibitor constant K i .
- the K i describes that inhibitor concentration in which the activity is only 50% of the activity determined in the absence of the inhibitor.
- PGD enzymes according to the invention can, due to their feedback resistance for the production of amino acids of the phosphoglycerate family or of compounds resulting from the C1 metabolism derive, be used.
- inventive serA alleles expressed in a host strain are used.
- a serA allele according to the invention can be described under Control of the own promoter localized in front of the serA gene or by using other suitable promoter systems, which are known in the art, take place. This can be the corresponding allele, for example, under the control of a such promoter in either one or more copies located on the chromosome of the host organism.
- the strategies for the integration of genes into the chromosome are state of the art Technology.
- the serA allele to be expressed is preferred cloned into a vector, preferably a plasmid.
- the invention therefore also relates to a vector, characterized that he under a serA allelic according to the invention functional control of a promoter.
- vectors which already contain genetic elements (e.g. constitutive or regulatable promoters, terminators), either an ongoing or a controlled, allow inducible expression of the gene coding for the PGD. They are also located on an expression vector preferably other regulatory elements such as ribosomal Binding sites and termination sequences as well as sequences that code for selective markers and / or reporter genes.
- the expression Such selection marker facilitates identification of transformants. Suitable as selection markers Genes that are resistant to z. B. ampicillin, Tetracycline, chloramphenicol, kanamycin or other antibiotics encode.
- the plasmid vector should preferably contain an origin point of replication.
- plasmid vectors such as the E. coli vectors pACYC184, pUC18, pQE-70, pBR322, pSC101 and their derivatives.
- Suitable inducible promoters are, for example the lac, tac, trc, lambda PL, ara or tet promoter or sequences derived therefrom.
- Such vectors allow the direct production of inventive Microorganism strains with high production capacity from any microorganism strain, as such Plasmid also the lifting of other restrictions of the Metabolic pathway in a microorganism causes.
- a common transformation method e.g., electroporation
- the serA allele-containing plasmids according to the invention incorporated in microorganisms and, for example, by antibiotic resistance selected for plasmid-carrying clones.
- the invention thus also relates to processes for the preparation a microorganism strain according to the invention, characterized that in a microorganism strain an inventive Vector is introduced.
- a host organism for vectors of the invention all organisms suitable for the biosynthetic pathway for amino acids the phosphoglycerate family, recombinant Process are accessible and cultivable by fermentation are.
- Such microorganisms can be fungi, yeasts or bacteria be. Preference is given to bacteria of the phylogenetic group Eubacteria are used. Particularly preferred are microorganisms the family Enterobacteriaceae and in particular the Species Escherichia coli.
- the invention thus further relates to a microorganism strain, for the fermentative production of amino acids of Phosphoglycerate family or their derivatives or compounds, which derive from the C1 metabolism suitable is, characterized in that it is a PGD according to the invention has.
- Another object of the invention is the production of Amino acids of the phosphoglycerate family or of compounds, which derive from the C1 metabolism, by cultivation a microorganism strain according to the invention.
- the microorganism strain according to the invention for example cultured in a fermenter in a nutrient medium, the a suitable carbon, and a suitable source of energy, and other additives.
- the substances formed during the fermentation such as L-phosphoserine, L-serine, O-acetyl-L-serine, L-cysteine, Glycine, L-tryptophan, 1,2,4-triazol-2-yl-L-alanine, L-methionine or pantothenic acid can subsequently be purified become.
- the serA gene from Escherichia coli strain W3110 was amplified by the polymerase chain reaction.
- the oligonucleotides were used as specific primers and
- the resulting DNA fragment was digested with the restriction enzymes NdeI and HindIII digested and the 5 'overhangs with Klenow enzyme refilled. Subsequently, the DNA fragment was using purified by agarose gel electrophoresis and using the GeneClean method isolated (GeneClean Kit BI0101 P.O.Box 2284 La Jolla, California, 92038-2284). The cloning of the thus obtained serA fragment was inserted into the expression vector pQE-70 (Qiagen, Hilden, D). For this, the vector was first with SphI and BamHI cut and the 3 'overhang with Klenow enzyme or the 5 'overhang filled with Klenow enzyme.
- the vector fragment was purified and washed with ligated to the serA fragment.
- the resulting vector carries the Designation pFL209.
- the bacterial strain Escherichia coli JM109 / pFL209 was used in the DSMZ (German Collection for microorganisms and cell cultures GmbH, D-38142 Braunschweig) under the number DSM 15628 according to the Budapest Treaty deposited.
- Site-directed mutagenesis at codons 349 and 372 of the serA gene was performed by an inverse polymerase chain reaction.
- the template used was the vector pFL209 described in Example 1.
- the primers were and used.
- 100 ml LB medium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l NaCl), which additionally contained 100 mg / l ampicillin , inoculated with a 2 ml overnight culture of the strains with the plasmid-coded serA alleles and incubated at 30 ° C and 150 rpm in a shaker.
- serA expression was induced by the addition of 0.4 mM isopropyl- ⁇ -thiogalactoside, and the culture was incubated for a further 3 hours.
- the cells were then harvested by centrifugation, washed and resuspended in 2 ml of buffer (100 mM K-phosphate pH 7.0, 10 mM MgCl 2 , 1 mM dithiothreitol). Cell disruption was performed using a French Press (Spectronic Instruments, Inc., Rochester, NY, USA) at a pressure of 18,000 psi. The crude extracts were clarified by centrifugation at 30,000 g and PGD activity was determined by the test of McKrickick and Pizer (1980, J. Bacteriol 141: 235-245). The following tables show the PGD activity of different mutants, as well as the corresponding inhibitor constants K i .
- a combination of codons 349 and 372 mutations should show whether the exchanges have a synergistic effect on feedback resistance.
- a unique HindII restriction site between the two mutation sites was used.
- HindIII-HindIII restriction of the serA20 allele vector isolated a 183 bp fragment corresponding to the 3 'end of the serA gene and containing the mutation at codon 372. This fragment was cloned into a similarly Hindllam-BamHI digested vector with the serA40 allele to give a clone which is a double mutant.
- the following table shows the associated enzyme data. Mutations at codons 349 and 372 allele mutation Activity [units / mg] Ki [mM] serA wildtype 0.05 ⁇ 0.1 serA2040 G349D, T372I 0.05 120
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Abstract
Description
Die Erfindung betrifft Varianten der 3-Phospoglyceratdehydrogenase mit reduzierter Hemmung durch L-Serin und dafür codierende Gene.The invention relates to variants of 3-phosphoglycerate dehydrogenase with reduced inhibition by L-serine and coding for it Genes.
Die Herstellung der zwanzig natürlichen, proteinogenen Aminosäuren wird heutzutage vorwiegend durch Fermentation von Mikroorganismen bewerkstelligt. Dabei wird ausgenützt, dass Mikroorganismen über entsprechende Biosynthesewege zur Synthese der natürlichen Aminosäuren verfügen.The production of the twenty natural, proteinogenic amino acids Nowadays, it is predominantly fermented by microorganisms accomplished. It exploits that microorganisms via appropriate biosynthetic pathways for synthesis of natural amino acids.
Solche Biosynthesewege unterliegen jedoch in Wildtyp-Stämmen einer strengen Kontrolle, die gewährleistet, dass die Aminosäuren nur zum Eigenbedarf der Zelle hergestellt werden. Ein wichtiger Kontrollmechanismus in vielen Biosynthesen ist beispielsweise das Phänomen der Feedback-Hemmung (oder Endprodukt-Hemmung). Dabei wird meist dasjenige Enzym eines Biosyntheseweges, das die einleitende enzymatische Reaktion dieses Biosynthesewegs katalysiert, durch das Endprodukt des Biosyntheseweges gehemmt. Die Hemmung findet meist durch eine allosterische Bindung des Endproduktes an das Enzym statt und bewirkt eine Konformationsänderung in einen inaktiven Zustand. So wird sichergestellt, dass bei einer Anhäufung des Endproduktes in der Zelle die weitere Synthese durch Hemmung des einleitenden Schrittes unterbunden wird.However, such biosynthetic pathways are subject to wild-type strains a strict control that ensures that the amino acids be made only for personal use of the cell. One For example, important control mechanism in many biosyntheses the phenomenon of feedback inhibition (or end product inhibition). Usually the enzyme of a biosynthetic pathway, this is the preliminary enzymatic reaction of this Biosynthesis pathways catalyzed by the end product of Biosyntheseweges inhibited. The inhibition usually takes place through an allosteric Binding of the final product to the enzyme instead of and causes a conformational change to an inactive state. This ensures that when an accumulation of the final product in the cell further synthesis by inhibition of introductory step is prevented.
Eine effiziente industrielle Produktion von Stoffwechselprodukten (wie z.B. Aminosäuren) ist deshalb nur möglich, wenn die Restriktionen durch Feedbackhemmung eines Stoffwechselweges aufgehoben werden können und damit Mikroorganismen verfügbar gemacht werden, die im Gegensatz zu Wildtyp-Organismen eine drastisch gesteigerte Produktionsleistung für die Herstellung des gewünschten Stoffwechselprodukts aufweisen.An efficient industrial production of metabolic products (such as amino acids) is therefore only possible if the restrictions by feedback inhibition of a metabolic pathway can be repealed and thus microorganisms available which, in contrast to wild-type organisms, a drastically increased production capacity for production have the desired metabolite.
Die Phosphoglycerat-Familie von Aminosäuren ist dadurch definiert, dass es sich um Aminosäuren handelt, die in ihrer Biosynthese von der 3-Phosphoglycerinsäure abgeleitet werden. Der natürliche Pfad des Stoffwechsels führt dabei zunächst über die Zwischenstufen 3-Phospohydroxypyruvat und 3-Phospho-Lserin zu L-Serin. L-Serin kann weiterhin zu Glycin bzw. über O-Acetyl-Serin zu L-Cystein umgesetzt werden. Auch L-Tryptophan ist zu dieser Gruppe zu zählen, da es sich ebenfalls in der Biosynthese von L-Serin ableitet. Ebenso sind unnatürliche Aminosäuren, die nach dem in US 2002/0039767 A1 beschrieben Verfahren hergestellt werden, der Phosphoglycerat-Familie zuzuordnen.The phosphoglycerate family of amino acids is defined by that they are amino acids involved in their biosynthesis derived from the 3-phosphoglyceric acid. Of the natural path of metabolism leads to it over the intermediates 3-Phospohydroxypyruvat and 3-phospho-Lserin to L-serine. L-serine can continue to glycine or over O-acetyl-serine be converted to L-cysteine. Also L-tryptophan is to be counted to this group, since it is also in derived from the biosynthesis of L-serine. Likewise, unnatural Amino acids described in US 2002/0039767 A1 Process to be assigned to the phosphoglycerate family.
Verbindungen, die sich aus dem C1-Stoffwechsel ableiten, zeigen ebenfalls eine Abhängigkeit von der Biosynthese der Aminosäuren der Phosphoglycerat-Familie. Dies beruht auf der Tatsache, dass bei der Konversion von L-Serin zu Glycin Tetrahydrofolat als C1-Gruppenakzeptor fungiert und das beladende Tetrahydrofolat als zentraler Methylgruppendonor im C1-Stoffwechsel an vielen Biosynthesen (z.B. L-Methionin, Nukleotide, Pantothensäure, etc.) beteiligt ist. Erfindungsgemäß sind Verbindungen, die sich aus dem C1-Stoffwechsel ableiten somit vorzugsweise Verbindungen, die in ihrer Biosynthese von einer C1-Gruppenübertragung über Tetrahydrofolsäure abhängen.Compounds derived from the C1 metabolism show also a dependence on the biosynthesis of the amino acids the phosphoglycerate family. This is based on the fact that in the conversion of L-serine to glycine tetrahydrofolate acts as a C1 group acceptor and the loading tetrahydrofolate as a central methyl group donor in C1 metabolism in many biosyntheses (e.g., L-methionine, nucleotides, pantothenic acid, etc.). According to the invention, compounds derive from the C1 metabolism thus preferably Compounds that in their biosynthesis of a C1 group transfer depend on tetrahydrofolic acid.
Der einleitende Schritt der Biosynthese von Aminosäuren der Phosphoglycerat-Familie ist die Oxidation der D-3-Phosphoglycerinsäure zu 3-Phosphohydroxypyruvat und wird durch das Enzym 3-Phosphoglyceratdehydrogenase (PGD) [EC 1.1.1.95] katalysiert. Als Akzeptor für die bei der Reaktion entstehenden Reduktionsäquivalente dient NAD+, das zu NADH/H+ umgesetzt wird.The initial step in the biosynthesis of amino acids of the phosphoglycerate family is the oxidation of D-3-phosphoglyceric acid to 3-phosphohydroxypyruvate and is catalyzed by the enzyme 3-phosphoglycerate dehydrogenase (PGD) [EC 1.1.1.95]. NAD + , which is converted into NADH / H + , serves as the acceptor for the reduction equivalents formed during the reaction.
PGD-Enzyme sind aus verschiedensten Organismen bekannt (z.B. Rattus norvegicus, Arabidopsis thaliana, Escherichia coli, Bacillus subtilis). Dabei unterliegen die besser charakterisierten mikrobiellen Vertreter einer Feedback-Hemmung durch L-Serin. PGD enzymes are known from a variety of organisms (e.g. Rattus norvegicus, Arabidopsis thaliana, Escherichia coli, Bacillus subtilis). The better characterized microbial representatives of feedback inhibition by L-serine.
Auf der Ebene der Aminosäuresequenz sind die mikrobiellen PGD-Enzyme im N-terminalen Teil (Aminosäure 1-340 der E. coli Sequenz) untereinander sehr ähnlich, wogegen der C-terminale Anteil nur geringe Ähnlichkeiten aufweist. Gerade in diesem C-terminalen Teil ist die regulatorische Domäne lokalisiert, die für die Serin-Hemmung verantwortlich ist (Peters-Wendisch et al., 2002, Appl. Microbiol. Biotechnol. 60:437-441).At the level of the amino acid sequence are the microbial PGD enzymes in the N-terminal part (amino acid 1-340 of the E. coli sequence) very similar to each other, whereas the C-terminal portion has little similarity. Especially in this C-terminal Part is localized to the regulatory domain, the is responsible for serine inhibition (Peters-Wendisch et al., 2002, Appl. Microbiol. Biotechnol. 60: 437-441).
Die am besten charakterisierte PGD ist diejenige aus Escherichia coli. Das Enzym wurde eingehend biochemisch untersucht (Dubrow & Pizer, 1977, J. Biol. Chem. 252, 1527-1538) und unterliegt einer allosterischen Feedback-Hemmung durch L-Serin mit einer Inhibitorkonstante Ki von 5 µM.The best characterized PGD is that of Escherichia coli. The enzyme has been extensively biochemically studied (Dubrow & Pizer, 1977, J. Biol. Chem. 252, 1527-1538) and is subject to allosteric feedback inhibition by L-serine with an inhibitor constant K i of 5 μM.
Diese Feedback-Hemmung steht einer effizienten Produktion von Aminosäuren der Phosphoglycerat-Familie im Wege und war deshalb bereits Ziel molekularbiologischer Ansätze.This feedback inhibition stands for an efficient production of Amino acids of the phosphoglycerate family in the way and was therefore already the goal of molecular biological approaches.
So wurden in der Schrift EP0620853A Varianten der Escherichia coli PGD mit verringerter Empfindlichkeit gegen Hemmung durch Serin beschrieben, die eine Modifikation in den C-terminalen 25% der Wildtyp-PGD, (d.h. Aminosäuren 307-410), bevorzugt eine Modifikation im Bereich der letzten 50 Reste (d.h. Aminosäuren 361-410) aufweisen. Die beschriebenen Mutanten wurden durch Linker-Mutagenese, d.h. durch einfache Nutzung von vorhandenen Restriktionsschnittstellen im E. coli serA-Gen und anschließendes Einsetzen von Oligonukleotid-Linkern einer Länge von 8-14 Basenpaaren erhalten.Thus, in the document EP0620853A variants of Escherichia coli PGD with reduced sensitivity to inhibition by Serine described a modification in the C-terminal 25% of the wild-type PGD (i.e., amino acids 307-410), preferably one Modification in the region of the last 50 residues (i.e., amino acids 361-410). The described mutants were by linker mutagenesis, i. by easy use of existing ones Restriction interfaces in the E. coli serA gene and subsequent insertion of oligonucleotide linkers of a length of 8-14 base pairs.
Solche Linker-Mutagenesen sind jedoch meist problematisch, da durch den Einbau bzw. die Deletion von mehreren Resten die Struktur des Proteins sehr stark verändert wird und so die Gesamtaktivität oder Stabilität des Proteins negativ beeinflusst wird. Tatsächlich zeigen die meisten in EP0620853A beschriebenen Mutanten eine kaum nachweisbare Aktivität. However, such linker mutagenesis are usually problematic because by the incorporation or deletion of several residues the Structure of the protein is very much changed and so the overall activity or stability of the protein adversely affected becomes. In fact, most of them are described in EP0620853A Mutants a barely detectable activity.
Auch in coryneformen Mikroorganismen wurden Mutagenesen am serA-Gen vorgenommen, die das Ziel einer Verringerung der Empfindlichkeit der PGD gegen Hemmung durch Serin erfüllen:
- Peters-Wendisch et al. (2002, Appl. Microbiol. Biotechnol. 60:437-441) beschreiben C-terminale Deletionen der PGD von Corynebacterium glutamicum. Auch hier führen die Deletionen zu teilweise starken Einbußen an Enzymaktivität.
- Die Anmeldung EP0943687A2 beschreibt einen Austausch des Glutaminsäure-Restes an Position 325 der PGD von Brevibacterium flavum. Dieser Rest liegt im Bezug zur Escherichia coli PGD in einem Alignment mit dem Algorithmus GAP des Programms GCG (GCG Wisconsin Package, Genetics Computer Group (GCG) Madison, Wisconsin) bereits in dem variablen C-terminalen Teil des Proteins und korreliert mit dem Asparaginrest 364 des Escherichia coli Proteins. Da diese Modifikation in dem variablen C-terminalen Teil der PGD liegen, sind keine Rückschlüsse auf das Escherichia coli Protein möglich.
- Peters-Wendisch et al. (2002, Appl.Microbiol.Biotechnol 60: 437-441) describe C-terminal deletions of PGD of Corynebacterium glutamicum. Again, the deletions lead to some strong losses of enzyme activity.
- The application EP0943687A2 describes an exchange of the glutamic acid residue at position 325 of the PGD of Brevibacterium flavum. This residue is already in the variable C-terminal part of the protein and correlates with the asparagine residue 364 in an alignment with the GAP algorithm of the GCG program (GCG Wisconsin Package, Genetics Computer Group (GCG) Madison, Wisconsin) in relation to Escherichia coli PGD of the Escherichia coli protein. Since these modifications are in the variable C-terminal part of the PGD, no conclusions are possible on the Escherichia coli protein.
Aufgabe der vorliegenden Erfindung war es, Varianten der PGD von Escherichia coli zur Verfügung zu stellen, die eine im Vergleich zur Escherichia coli-Wildtyp-PGD verringerte Empfindlichkeit gegen eine Hemmung durch Serin aufweisen.Object of the present invention was to variants of PGD of Escherichia coli which have an im Compared to Escherichia coli wild-type PGD decreased sensitivity against inhibition by serine.
Die Aufgabe wird gelöst durch eine PGD mit einer Aminosäuresequenz, die dadurch gekennzeichnet ist, dass sie sich von der Aminosäuresequenz der Escherichia coli Wildtyp PGD (SEQ ID NO: 2) mit einem Methionin als Position 1 dadurch unterscheidet, dass an Position 349 eine Aminosäure mit Ausnahme von Glycin oder an Position 372 eine Aminosäure mit Ausnahme von Threonin befindet.The object is achieved by a PGD having an amino acid sequence, which is characterized by being different from the Amino acid sequence of Escherichia coli wild-type PGD (SEQ ID NO: 2) with a methionine as position 1 differs thereby that at position 349 an amino acid except glycine or at position 372 an amino acid except threonine located.
Eine erfindungsgemäße PGD kann auch Mutationen an beiden genannten Positionen von SEQ ID NO: 1 aufweisen.A PGD according to the invention may also have mutations in both Have positions of SEQ ID NO: 1.
Die Erfindung betrifft ferner eine DNS-Sequenz codierend für eine erfindungsgemäße PGD. Dieses serA-Allel unterscheidet sich vom Gen der Escherichia coli PGD (serA-Gen, SEQ ID NO: 1) dadurch, dass das Codon 349 für eine natürliche Aminosäure mit Ausnahme von Glycin oder das Codon 372 für eine natürliche Aminosäure mit Ausnahme von Threonin codiert.The invention further relates to a DNA sequence coding for a PGD according to the invention. This serA allele is different different from the gene of Escherichia coli PGD (serA gene, SEQ ID NO: 1) in that the codon 349 is a natural amino acid Exception of glycine or codon 372 for a natural amino acid coded with the exception of threonine.
Ein erfindungsgemäßes serA-Allel kann auch eine Mutation an beiden genannten Codons aufweisen.A serA allele according to the invention may also have a mutation have two codons mentioned.
Im Rahmen der vorliegenden Erfindung sind als erfindungsgemäße serA-Allele auch solche Gene aufzufassen, die bei einer Analyse mit dem Algorithmus GAP (GCG Wisconsin Package, Genetics Computer Group (GCG) Madison, Wisconsin) eine Sequenzidentität von größer 30 % aufweisen, sofern sie eine der genannten Mutationen aufweisen. Besonders bevorzugt ist eine Sequenzidentität von größer 70 %.In the context of the present invention are as inventive serA alleles include such genes that are analyzed with the algorithm GAP (GCG Wisconsin Package, Genetics Computer Group (GCG) Madison, Wisconsin) a sequence identity greater than 30% if they have any of the above mutations exhibit. Particularly preferred is a sequence identity greater than 70%.
Ebenso sind Proteine mit einer Sequenzidentität von größer 40 % ermittelt mit dem Algorithmus GAP, im Sinne der vorliegenden Erfindung als von der E. coli PGD abgeleitete Proteine aufzufassen, sofern sie eine PGD Aktivität und einen der genannten Aminosäureaustausche aufweisen. Besonders bevorzugt ist eine Sequenzidentität von größer 70 %.Likewise, proteins with a sequence identity greater than 40 % determined with the algorithm GAP, in the sense of the present To conceive of the invention as proteins derived from E. coli PGD, provided they have a PGD activity and one of the above Have amino acid substitutions. Particularly preferred is a Sequence identity greater than 70%.
Ferner sind als erfindungsgemäße Gene Allelvarianten des serA-Gens zu verstehen, die sich durch Deletion, Insertion oder Substitution von Nukleotiden aus der in SEQ ID NO: 1 dargestellten Sequenz ableiten, wobei die enzymatische Aktivität des Genprodukts mehr als 10 % der Aktivität des Wildtyp Genprodukts entspricht und eine Mutation des Codons 349 für die Aminosäure Glycin bzw. des Codons 372 für die Aminosäure Threonin, oder eine Kombination der beiden Mutationen vorliegt.Furthermore, genes according to the invention are allelic variants of the serA gene to be understood by deletion, insertion or Substitution of nucleotides from that shown in SEQ ID NO: 1 Derive sequence, with the enzymatic activity of the gene product more than 10% of the activity of the wild-type gene product and a mutation of codon 349 for the Amino acid glycine or codon 372 for the amino acid threonine, or a combination of the two mutations.
PGD-Varianten, die einen Aminosäureaustausch an Position 349 oder an Position 372 oder eine Kombination davon besitzen, können mit Standardtechniken der Molekularbiologie erzeugt werden. Dazu werden an den entsprechenden Codons Mutationen in das für die PGD codierende serA-Gen eingebracht. Entsprechende Methoden zur Einführung von Mutationen an spezifischen Positionen innerhalb eines DNS-Fragmentes sind bekannt. PGD variants that have an amino acid substitution at position 349 or at position 372 or a combination thereof, can be generated using standard techniques of molecular biology become. For this purpose, mutations are made in the corresponding codons introduced the serA gene encoding the PGD. Appropriate Methods for introducing mutations at specific positions within a DNA fragment are known.
Als Ausgangsmaterial für die Mutagenese dient vorzugsweise die DNS des E. coli serA-Gens. Das zu mutierende serA-Gen kann chromosomal oder extrachromosomal codiert sein. Bevorzugt wird das serA-Gen jedoch durch Polymerase-Ketten-Reaktion amplifiziert und in einen Vektor kloniert. Durch Anwendung der vorgenannten Mutagenese-Methoden werden ein oder mehrere Nukleotide der DNS-Sequenz so verändert, dass die codierte PGD einen Aminosäureaustausch an Position 349 oder 372 aufweist, wobei Position 1 das Startmethionin aus SEQ ID NO: 1 ist.The starting material for the mutagenesis is preferably the DNA of the E. coli serA gene. The serA gene to be mutated can chromosomally or extrachromosomally encoded. It is preferred However, the serA gene amplified by polymerase chain reaction and cloned into a vector. By applying the aforementioned Mutagenesis methods are one or more nucleotides the DNA sequence is altered so that the encoded PGD undergoes an amino acid exchange at position 349 or 372, wherein position 1 is the starting methionine of SEQ ID NO: 1.
Diese Mutationen bewirken, dass die codierte PGD eine verminderte Empfindlichkeit gegen Hemmung durch L-Serin (=Feedback-Resistenz) besitzt. Dabei ist besonders vorteilhaft, dass die erfindungsgemäßen PGD-Varianten in Abwesenheit von L-Serin im Vergleich zur Wildtyp-PGD eine Aktivität von mehr als 10 %, vorzugsweise eine unveränderte Aktivität, aufweist.These mutations cause the encoded PGD to diminish one Sensitivity to inhibition by L-serine (= feedback resistance) has. It is particularly advantageous that the PGD variants according to the invention in the absence of L-serine in An activity of more than 10% compared to wild-type PGD, preferably an unchanged activity.
Zur Bestimmung des Ausmaßes der Feedback-Resistenz einer erfindungsgemäßen PGD-Variante kann jede Methode benützt werden, die es erlaubt, die Aktivität des Enzyms in Anwesenheit von L-Serin zu bestimmen. Beispielsweise kann die Bestimmung der PGD-Aktivität in Anlehnung an die von McKitrick und Pizer (1980, J.Bacteriol. 141:235-245) beschriebene Methode erfolgen. Die Enzymaktivität wird anhand der Rückreaktion in einem Ansatz, der Phosphohydroxypyruvat und NADH/H+ enthält, gemessen. Die Reaktion wird durch Enzymzugabe gestartet und über die Abnahme der Extinktion bei 340 nm, die durch Oxidation des NADH/H+ hervorgerufen wird, in einem Spektralphotometer verfolgt. Die Hemmung der PGD-Aktivität wird in Anwesenheit verschiedener Konzentrationen von L-Serin im Reaktionsansatz getestet. Die katalytische Aktivität der verschiedenen PGD-Varianten wird in An- und Abwesenheit von L-Serin bestimmt und daraus die Inhibitorkonstante Ki ermittelt. Der Ki beschreibt diejenige Inhibitorkonzentration, bei welcher die Aktivität nur noch 50 % der Aktivität beträgt, die in Abwesenheit des Inhibitors bestimmt wurde. To determine the extent of feedback resistance of a PGD variant according to the invention, any method can be used which allows to determine the activity of the enzyme in the presence of L-serine. For example, the determination of PGD activity can be based on the method described by McKrickick and Pizer (1980, J. Bacteriol 141: 235-245). The enzyme activity is measured by the reverse reaction in an approach containing phosphohydroxypyruvate and NADH / H + . The reaction is started by enzyme addition and monitored by the decrease in absorbance at 340 nm, which is caused by oxidation of NADH / H + in a spectrophotometer. The inhibition of PGD activity is tested in the presence of various concentrations of L-serine in the reaction mixture. The catalytic activity of the various PGD variants is determined in the presence and absence of L-serine and used to determine the inhibitor constant K i . The K i describes that inhibitor concentration in which the activity is only 50% of the activity determined in the absence of the inhibitor.
Erfindungsgemäße PGD-Enzyme können aufgrund ihrer Feedback-Resistenz zur Herstellung von Aminosäuren der Phosphoglycerat-Familie oder von Verbindungen, die sich aus dem C1-Stoffwechsel ableiten, verwendet werden. Hierfür werden die erfindungsgemäßen serA-Allele in einem Wirtsstamm exprimiert.PGD enzymes according to the invention can, due to their feedback resistance for the production of amino acids of the phosphoglycerate family or of compounds resulting from the C1 metabolism derive, be used. For this purpose, the inventive serA alleles expressed in a host strain.
Die Expression eines erfindungsgemäßen serA-Allels kann unter Kontrolle des eigenen, vor dem serA-Gen lokalisierten Promotors oder durch Verwendung anderer geeigneter Promotorsysteme, die dem Fachmann bekannt sind, erfolgen. Dabei kann sich das entsprechende Allel beispielsweise unter der Kontrolle eines solchen Promotors entweder in einer oder in mehreren Kopien auf dem Chromosom des Wirtsorganismus befinden. Die Strategien zur Integration von Genen in das Chromosom sind Stand der Technik. Bevorzugt wird das zu exprimierende serA-Allel jedoch in einen Vektor kloniert, vorzugsweise ein Plasmid.The expression of a serA allele according to the invention can be described under Control of the own promoter localized in front of the serA gene or by using other suitable promoter systems, which are known in the art, take place. This can be the corresponding allele, for example, under the control of a such promoter in either one or more copies located on the chromosome of the host organism. The strategies for the integration of genes into the chromosome are state of the art Technology. However, the serA allele to be expressed is preferred cloned into a vector, preferably a plasmid.
Die Erfindung betrifft daher auch einen Vektor, dadurch gekennzeichnet, dass er ein erfindungsgemäßes serA-Allel unter funktioneller Kontrolle eines Promotors enthält.The invention therefore also relates to a vector, characterized that he under a serA allelic according to the invention functional control of a promoter.
Zur Klonierung der erfindungsgemäßen serA-Allele können Vektoren verwendet werden, die bereits genetische Elemente (z.B. konstitutive oder regulierbare Promotoren, Terminatoren) enthalten, die entweder eine andauernde oder eine kontrollierte, induzierbare Expression des für die PGD codierenden Gens ermöglichen. Außerdem befinden sich auf einem Expressionsvektor vorzugsweise andere regulatorische Elemente wie ribosomale Bindungsstellen und Terminationssequenzen sowie Sequenzen, die für selektive Marker und/oder Reporter-Gene codieren. Die Expression derartiger Selektionsmarker erleichtert die Identifizierung von Transformanten. Als Selektionsmarker geeignet sind Gene, die für eine Resistenz gegenüber z. B. Ampicillin, Tetracyclin, Chloramphenicol, Kanamycin oder anderen Antibiotika codieren. Wenn das erfindungsgemäße serA-Allel extrachromosomal repliziert werden soll, sollte der Plasmidvektor vorzugsweise einen Ursprungspunkt der Replikation enthalten. Besonders bevorzugt sind Plasmidvektoren wie beispielsweise die E. coli-Vektoren pACYC184, pUC18, pQE-70, pBR322, pSC101 und ihre Derivate. Als induzierbare Promotoren eignen sich beispielsweise der lac-, tac-, trc-, lambda PL, ara- oder tet-Promotor oder davon abgeleitete Sequenzen.For cloning the serA alleles according to the invention, vectors which already contain genetic elements (e.g. constitutive or regulatable promoters, terminators), either an ongoing or a controlled, allow inducible expression of the gene coding for the PGD. They are also located on an expression vector preferably other regulatory elements such as ribosomal Binding sites and termination sequences as well as sequences that code for selective markers and / or reporter genes. The expression Such selection marker facilitates identification of transformants. Suitable as selection markers Genes that are resistant to z. B. ampicillin, Tetracycline, chloramphenicol, kanamycin or other antibiotics encode. When the serA allele according to the invention is extrachromosomal should be replicated, the plasmid vector should preferably contain an origin point of replication. Especially preferred are plasmid vectors such as the E. coli vectors pACYC184, pUC18, pQE-70, pBR322, pSC101 and their derivatives. Suitable inducible promoters are, for example the lac, tac, trc, lambda PL, ara or tet promoter or sequences derived therefrom.
Des weiteren sind Plasmidvektoren besonders bevorzugt, die bereits ein Gen/Allel enthalten, dessen Einsatz ebenfalls zu einer Überproduktion von Aminosäuren der Phosphoglycerat-Familie bzw. von Verbindungen, die sich aus dem C1-Stoffwechsel ableiten, führt, wie beispielsweise für die Produktion von:
- L-Serin (z.B. serB-, serC-, Exportcarrier-Gen wie beschreiben in DE10044831A1)
- N-Acetyl-Serin, O-Acetyl-Serin, Cystin, Cystein oder Cysteinderivaten (z.B. cysE-Allele wie beschrieben in WO97/15673, Efflux-Gene wie beschrieben in EP0885962A1, cysB-Gen wie beschrieben in DE19949579C1, yfiK-Gen wie beschrieben in DE 10232930A)
- L-Tryptophan (z.B. trpE-Allele wie beschrieben in EP0662143A)
- Pantothensäure (z.B. wie beschrieben in WO02061108)
- L-serine (eg serB, serC, export carrier gene as described in DE10044831A1)
- N-acetyl-serine, O-acetyl-serine, cystine, cysteine or cysteine derivatives (eg cysE alleles as described in WO97 / 15673, efflux genes as described in EP0885962A1, cysB gene as described in DE19949579C1, yfiK gene as described in DE 10232930A)
- L-tryptophan (eg trpE alleles as described in EP0662143A)
- Pantothenic acid (eg as described in WO02061108)
Solche Vektoren ermöglichen die direkte Herstellung von erfindungsgemäßen Mikroorganismenstämmen mit hoher Produktionsleistung aus einem beliebigen Mikroorganismenstamm, da ein solches Plasmid auch die Aufhebung von anderen Restriktionen des Stoffwechselweges in einem Mikroorganismus bewirkt.Such vectors allow the direct production of inventive Microorganism strains with high production capacity from any microorganism strain, as such Plasmid also the lifting of other restrictions of the Metabolic pathway in a microorganism causes.
Durch eine gängige Transformationsmethode (z.B. Elektroporation) werden die erfindungsgemäßen serA-Allel-haltigen Plasmide in Mikroorganismen eingebracht und beispielsweise mittels Antibiotika-Resistenz auf plasmid-tragende Klone selektiert.By a common transformation method (e.g., electroporation) become the serA allele-containing plasmids according to the invention incorporated in microorganisms and, for example, by antibiotic resistance selected for plasmid-carrying clones.
Die Erfindung betrifft somit auch Verfahren zur Herstellung eines erfindungsgemäßen Mikroorganismenstammes, dadurch gekennzeichnet, dass in einen Mikroorganismenstamm ein erfindungsgemäßer Vektor eingebracht wird. The invention thus also relates to processes for the preparation a microorganism strain according to the invention, characterized that in a microorganism strain an inventive Vector is introduced.
Es ist auch möglich Vektoren mit einem erfindungsgemäßen serA-Allel in Mikroorganismen einzubringen, die beispielsweise einzelne oder mehrere der oben genannten Gene/Allele bereits chromosomal exprimieren und bereits eine Überproduktion eines Stoffwechselprodukts aufweisen. In solchen Fällen kann durch das Einbringen eines erfindungsgemäßen serA-Allels die Produktionsleistung nochmals gesteigert werden.It is also possible vectors with a serA allele according to the invention in microorganisms, for example, single or more of the above genes / alleles already chromosomally express and already an overproduction of a Have metabolite. In such cases can through the introduction of a serA allele according to the invention the production output be increased again.
Generell sind als Wirtsorganismus für erfindungsgemäße Vektoren alle Organismen geeignet, die den Biosyntheseweg für Aminosäuren der Phosphoglycerat-Familie aufweisen, rekombinanten Verfahren zugänglich sind und durch Fermentation kultivierbar sind. Solche Mikroorganismen können Pilze, Hefen oder Bakterien sein. Bevorzugt kommen Bakterien der phylogenetischen Gruppe der Eubacteria zum Einsatz. Besonders bevorzugt sind Mikroorganismen der Familie Enterobacteriaceae und insbesondere der Art Escherichia coli.Generally, as a host organism for vectors of the invention all organisms suitable for the biosynthetic pathway for amino acids the phosphoglycerate family, recombinant Process are accessible and cultivable by fermentation are. Such microorganisms can be fungi, yeasts or bacteria be. Preference is given to bacteria of the phylogenetic group Eubacteria are used. Particularly preferred are microorganisms the family Enterobacteriaceae and in particular the Species Escherichia coli.
Die Erfindung betrifft somit ferner einen Mikroorganismenstamm, der zur fermentativen Herstellung von Aminosäuren der Phosphoglycerat-Familie oder deren Derivaten bzw. von Verbindungen, die sich aus dem C1 Stoffwechsel ableiten, geeignet ist, dadurch gekennzeichnet, dass er eine erfindungsgemäße PGD besitzt.The invention thus further relates to a microorganism strain, for the fermentative production of amino acids of Phosphoglycerate family or their derivatives or compounds, which derive from the C1 metabolism suitable is, characterized in that it is a PGD according to the invention has.
Ein weiterer Gegenstand der Erfindung ist die Herstellung von Aminosäuren der Phosphoglycerat-Familie bzw. von Verbindungen, die sich aus dem C1-Stoffwechsel ableiten, durch Kultivierung eines erfindungsgemäßen Mikroorganismenstammes.Another object of the invention is the production of Amino acids of the phosphoglycerate family or of compounds, which derive from the C1 metabolism, by cultivation a microorganism strain according to the invention.
Dazu wird der erfindungsgemäße Mikroorganismenstamm beispielsweise in einem Fermenter in einem Nährmedium kultiviert, das eine geeignete Kohlenstoff-, und eine geeignete Energiequelle, sowie andere Zusatzstoffe enthält.For this purpose, the microorganism strain according to the invention, for example cultured in a fermenter in a nutrient medium, the a suitable carbon, and a suitable source of energy, and other additives.
Die während der Fermentation gebildeten Substanzen wie beispielsweise L-Phosphoserin, L-Serin, O-Acetyl-L-Serin, L-Cystein, Glycin, L-Tryptophan, 1,2,4-Triazol-2-yl-L-alanin, L-Methionin oder Pantothensäure können anschließend aufgereinigt werden.The substances formed during the fermentation such as L-phosphoserine, L-serine, O-acetyl-L-serine, L-cysteine, Glycine, L-tryptophan, 1,2,4-triazol-2-yl-L-alanine, L-methionine or pantothenic acid can subsequently be purified become.
Die folgenden Beispiele dienen der weiteren Erläuterung der Erfindung. Sämtliche eingesetzten molekularbiologischen Verfahren, wie Polymerase-Kettenreaktion, Isolierung und Reinigung von DNS, Modifikation von DNS durch Restriktionsenzyme, Klenow-Fragment und Ligase, Transformation etc. wurden in der dem Fachmann bekannten, in der Literatur beschriebenen oder von den jeweiligen Herstellern empfohlenen Art und Weise durchgeführt.The following examples serve to further explain the Invention. All molecular biological procedures used, like polymerase chain reaction, isolation and purification DNA, modification of DNA by restriction enzymes, Klenow fragment and ligase, transformation, etc. were in the known in the art, described in the literature or recommended by the respective manufacturers way carried out.
Das serA-Gen aus Escherichia coli Stamm W3110 (American Type Culture Collection, ATCC27325) wurde mit Hilfe der Polymerase-Ketten-Reaktion amplifiziert. Als spezifische Primer dienten die Oligonukleotide und The serA gene from Escherichia coli strain W3110 (American Type Culture Collection, ATCC27325) was amplified by the polymerase chain reaction. The oligonucleotides were used as specific primers and
Das resultierende DNS-Fragment wurde mit den Restriktionsenzymen NdeI und HindIII verdaut und die 5'-Überhänge mit Klenow-Enzym aufgefüllt. Anschließend wurde das DNS-Fragment mit Hilfe einer Agarose-Gelelektrophorese gereinigt und mit der GeneClean-Methode isoliert (GeneClean Kit BI0101 P.O.Box 2284 La Jolla, California, 92038-2284). Die Klonierung des so erhaltenen serA-Fragments erfolgte in den Expressionsvektor pQE-70 (Qiagen, Hilden, D). Hierfür wurde der Vektor zunächst mit SphI und BamHI geschnitten und der 3'-Überhang mit Klenow-Enzym abgedaut bzw. der 5'-Überhang mit Klenow-Enzym aufgefüllt. Anschließend wurde das Vektorfragment gereinigt und mit dem serA-Fragment ligiert. Der resultierende Vektor trägt die Bezeichnung pFL209. Nach der Verifizierung des Konstrukts durch Sequenzierung wurde der Escherichia coli Stamm JM109 (Stratagene, Amsterdam, NL) transformiert und entsprechende Transformanten mit Ampicillin selektiert. Der Bakterienstamm Escherichia coli JM109 / pFL209 wurde bei der DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, D-38142 Braunschweig) unter der Nummer DSM 15628 gemäß Budapester Vertrag hinterlegt.The resulting DNA fragment was digested with the restriction enzymes NdeI and HindIII digested and the 5 'overhangs with Klenow enzyme refilled. Subsequently, the DNA fragment was using purified by agarose gel electrophoresis and using the GeneClean method isolated (GeneClean Kit BI0101 P.O.Box 2284 La Jolla, California, 92038-2284). The cloning of the thus obtained serA fragment was inserted into the expression vector pQE-70 (Qiagen, Hilden, D). For this, the vector was first with SphI and BamHI cut and the 3 'overhang with Klenow enzyme or the 5 'overhang filled with Klenow enzyme. Subsequently, the vector fragment was purified and washed with ligated to the serA fragment. The resulting vector carries the Designation pFL209. After verification of the construct by sequencing the Escherichia coli strain JM109 (Stratagene, Amsterdam, NL) and corresponding Transformants selected with ampicillin. The bacterial strain Escherichia coli JM109 / pFL209 was used in the DSMZ (German Collection for microorganisms and cell cultures GmbH, D-38142 Braunschweig) under the number DSM 15628 according to the Budapest Treaty deposited.
Die ortsspezifische Mutagenese an den Codons 349 und 372 des serA-Gens wurde mittels einer inversen Polymerase-Ketten-Reaktion durchgeführt. Als Matrize diente der in Beispiel 1 beschriebene Vektor pFL209. Für die Mutagenese des Codons 349 wurden die Primer und verwendet.Site-directed mutagenesis at codons 349 and 372 of the serA gene was performed by an inverse polymerase chain reaction. The template used was the vector pFL209 described in Example 1. For the mutagenesis of codon 349, the primers were and used.
Das erhaltene PCR-Produkt wurde durch Ligation zirkularisiert
und in den E. coli-Stamm JM109 transformiert. Durch Sequenzierung
wurde schließlich die Mutation an Codon 349 bestimmt und
die Korrektheit der übrigen Sequenz überprüft.
Für die Mutagenese des Codons 372 wurde prinzipiell gleich
verfahren, jedoch wurden die Primer
und
verwendet.The resulting PCR product was circularized by ligation and transformed into E. coli strain JM109. Sequencing finally determined the mutation at codon 349 and checked the correctness of the remaining sequence.
In principle the same procedure was followed for the mutagenesis of codon 372, but the primers became and used.
Zur Bestimmung von PGD-Enzymaktivitäten und des Einflusses von
L-Serin auf die Aktivität wurden 100 ml LB-Medium (10 g/l
Trypton, 5 g/l Hefeextrakt, 10 g/l NaCl), das zusätzlich 100
mg/l Ampicillin enthielt, mit einer 2 ml Übernachtkultur der
Stämme mit den plasmidcodierten serA-Allelen beimpft und bei
30 °C und 150 rpm in einem Schüttler inkubiert. Bei einer optischen
Dichte von 1,0 wurde die serA-Expression durch Zugabe
von 0,4 mM Isopropyl-β-thiogalaktosid induziert und die Kultur
für weitere 3 Stunden inkubiert. Die Zellen wurden anschließend
durch Zentrifugation geerntet, gewaschen und in 2 ml Puffer
(100 mM K-Phosphat pH 7,0; 10 mM MgCl2; 1 mM Dithiothreitol)
resuspendiert. Der Zellaufschluss erfolgte mittels einer
French Press (Spectronic Instruments, Inc., Rochester, NY,
USA) bei einem Druck von 18 000 psi. Die Rohextrakte wurden
durch Zentrifugation bei 30 000 g geklärt und die PGD-Aktivität
mit dem Test von McKitrick und Pizer (1980,
J.Bacteriol. 141:235-245) bestimmt.
Die folgenden Tabellen zeigen die PGD-Aktivität verschiedener
Mutanten, sowie die entsprechenden Inhibitorkonstanten Ki.
The following tables show the PGD activity of different mutants, as well as the corresponding inhibitor constants K i .
Eine Kombination von Mutationen des Codons 349 bzw. 372 sollte
zeigen, ob die Austausche einen synergistischen Effekt auf die
Feedback-Resistenz haben. Hierfür wurde eine singuläre HindII
Restriktionsschnittstelle zwischen den beiden Mutationsorten
benützt. So wurde durch HindII-HindIII-Restriktion des Vektors
mit dem serA20-Allel ein 183 bp-Fragment isoliert das dem 3'-Ende
des serA-Gens entspricht und die Mutation an Codon 372
beinhaltet. Dieses Fragment wurde in einen ebenfalls HindII-BamHI-verdauten
Vektor mit dem serA40-Allel kloniert und so
ein Klon erhalten der eine Doppelmutante darstellt. Die folgende
Tabelle zeigt die zugehörigen Enzymdaten.
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WO2007135188A2 (en) | 2006-05-24 | 2007-11-29 | Evonik Degussa Gmbh | Process for the preparation of l-methionine |
WO2010076324A1 (en) * | 2008-12-31 | 2010-07-08 | Metabolic Explorer | Method for the preparation of diols |
WO2012152664A1 (en) * | 2011-05-11 | 2012-11-15 | Wacker Chemie Ag | Method for producing l-cystine by fermentation under controlled oxygen saturation |
WO2013171098A3 (en) * | 2012-05-18 | 2014-01-09 | Wacker Chemie Ag | Method for producing l-cysteine using a benzoic acid-containing culture medium |
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WO2010076324A1 (en) * | 2008-12-31 | 2010-07-08 | Metabolic Explorer | Method for the preparation of diols |
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WO2012152664A1 (en) * | 2011-05-11 | 2012-11-15 | Wacker Chemie Ag | Method for producing l-cystine by fermentation under controlled oxygen saturation |
CN103517989A (en) * | 2011-05-11 | 2014-01-15 | 瓦克化学股份公司 | Method for producing l-cystine by fermentation under controlled oxygen saturation |
US9074230B2 (en) | 2011-05-11 | 2015-07-07 | Wacker Chemie Ag | Method for producing L-cystine by fermentation under controlled oxygen saturation |
CN103517989B (en) * | 2011-05-11 | 2015-11-25 | 瓦克化学股份公司 | By the method for fermentative production CYSTINE under in check oxygen saturation |
WO2013171098A3 (en) * | 2012-05-18 | 2014-01-09 | Wacker Chemie Ag | Method for producing l-cysteine using a benzoic acid-containing culture medium |
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US20050009162A1 (en) | 2005-01-13 |
EP1813669A1 (en) | 2007-08-01 |
DE502004009513D1 (en) | 2009-07-02 |
EP1950287A2 (en) | 2008-07-30 |
DE502004003661D1 (en) | 2007-06-14 |
ES2283906T3 (en) | 2007-11-01 |
EP1813669B1 (en) | 2009-05-20 |
DE502004009985D1 (en) | 2009-10-08 |
ATE440946T1 (en) | 2009-09-15 |
EP1496111B1 (en) | 2007-05-02 |
JP4695855B2 (en) | 2011-06-08 |
EP1950287B1 (en) | 2009-08-26 |
CN1609208A (en) | 2005-04-27 |
ES2325603T3 (en) | 2009-09-09 |
EP1496111A3 (en) | 2005-03-09 |
CN100526457C (en) | 2009-08-12 |
JP2005040134A (en) | 2005-02-17 |
ATE431847T1 (en) | 2009-06-15 |
EP1950287A3 (en) | 2008-08-13 |
US7582460B2 (en) | 2009-09-01 |
ATE361361T1 (en) | 2007-05-15 |
DE10331291A1 (en) | 2005-02-17 |
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